Developments in genetic engineering have made it possible to use genetic testing in applications such as virus detection, analysis of cell kinetics, predisposition testing, and drug response testing. RNA, which is one of the targets for genetic testing in these applications, is more unstable than DNA and can be easily degraded by endogenous ribonucleases contained in biological samples or when subjected to high-temperature/alkali treatment. Thus, high technologies, multi-step procedures, and expensive dedicated devices and reagents for preventing degradation of RNA have been required to prepare RNA.
For example, the following may be mentioned as known typical methods for recovering RNA from a biological sample: the acid guanidium-phenol-chloroform (AGPC) method (Non Patent Literature 1) which makes use of a combination of a protein denaturing agent and an organic solvent to dissolve the analyte and inactivate endogenous ribonucleases, thereby recovering the undegraded RNA; and the hot phenol method (Non Patent Literature 2). Unfortunately, both the methods are not only risky because of the use of an organic solvent and a high concentration of denaturing agent for inhibiting enzymatic reactions such as nucleic acid amplification, but also require a long-term, multi-step procedures to remove these materials, which is disadvantageous in terms of cost and ease of implementation.
Some techniques for easily preparing RNA have also been developed in which a strong chaotropic substance and a surfactant as protein denaturing agents are used to extract RNA, without using organic solvents, which extract is then directly subjected to an enzymatic reaction. For example, a method is known in which a biological sample is dissolved using guanidine thiocyanate and sarkosyl as denaturing agents, and then RNA is extracted while protecting RNA from degradation by endogenous ribonucleases (Patent Literature 1). The resulting extract can be directly subjected to an enzymatic reaction. These techniques facilitate the extraction of RNA compared to conventional techniques, by eliminating the need of steps for removing the denaturing agents before the extract is subjected to an enzymatic reaction. However, since the strong chaotropic substance, such as guanidine thiocyanate, and sarkosyl are strong protein denaturing agents, the presence of such strong denaturing agents in an enzymatic reaction system is undesirable in terms of efficiency of enzymatic reactions.
As a countermeasure to this, a nucleic acid extraction method that uses cholic acid or glycolic acid in order not to inhibit a subsequent enzymatic reaction has been known (Patent Literature 2). This technique eliminates the need of steps for purifying or diluting the nucleic acid extracted from a biological sample, and the nucleic acid can be directly subjected to an enzymatic reaction such as nucleic acid amplification. Unfortunately, this technique cannot prevent RNA degradation by endogenous ribonucleases and thus does not allow undegraded RNA to be extracted.